Mixed lineage leukemia histone methylases play critical
roles in estrogen-mediated regulation of HOXC13
Khairul I. Ansari, Sahba Kasiri, Imran Hussain and Subhrangsu S. Mandal
Department of Chemistry and Biochemistry, The University of Texas at Arlington, TX, USA
Introduction
Homeobox-containing genes are key players in
embryogenesis and development [1,2]. Misregulation of
homeobox genes is associated with tumorigenesis.
More than 200 homeobox-containing genes have been
identified in vertebrates, and they have been classified
into two major groups, class I and II. Class I homeo-
box-containing genes share a high degree of identity
(more than 80%) and are called HOX genes. In
humans, there are 39 different HOX genes, clustered
into four different groups, called HOXA, HOXB,
HOXC, and HOXD, located on chromosomes 7, 17,
12, and 2, respectively [1,2]. Each of these HOX genes
plays critical roles in embryogenesis and organo-
genesis. The nature of a body structure depends on the
specific combination of HOX gene products, and the
expression of specific HOX genes varies at different
stages of development. Therefore, proper regulation
and maintenance of HOX genes are essential for
normal physiological functions and growth.
HOXC13 is a critical gene involved in the regulation
of the hair keratin gene cluster and alopecia [3–5].
Transgenic mice overexpressing HOXC13 in differenti-
ating keratinocytes of hair follicles develop alopecia,
accompanied by a progressive pathological skin condi-
Keywords
estrogen; estrogen receptor; HOXC13 gene

mediated regulation of HOX genes are unknown. Herein, we have demon-
strated that MLLs are critical players in E2-dependent regulation of the
HOX gene.
Abbreviations
ChIP, chromatin immunoprecipitation; DEPC, diethyl pyrocarbonate; E2, estrogen (17b-estradiol); ER, estrogen receptor; ERE, estrogen-
response element; H3K4, histone H3 lysine 4; HMT, histone methyltransferase; MLL, mixed lineage leukemia histone methylase; NR,
nuclear receptor; RNAPII, RNA polymerase II.
7400 FEBS Journal 276 (2009) 7400–7411 ª 2009 The Authors Journal compilation ª 2009 FEBS
tion that resembles ichthyosis [4,5]. HOXC13 mutant
mice lack external hair, suggesting a critical role for
the gene in hair development [5]. HOXC13 has also
been found to be a fusion partner of NUP98 in adult
acute myeloid leukemia [6,7]. This protein also binds
to the ETS family transcription factor PU.1 and affects
the differentiation of murine erythroleukemia [8].
Although HOX13 is critical player in hair development
and disease, little is known about its own regulation.
Steroid hormones are critical players in sexual differen-
tiation. Steroid hormones such as estrogen (17b-estra-
diol; E2) and androgens are also linked with hair
follicle growth and differences in hair patterning
between males and females [9,10]. However, the molec-
ular mechanism of the roles of steroid hormones in
hair development is poorly understood. Herein, we
have investigated whether HOXC13, a critical player
in hair follicle development, is regulated by steroid
hormones.
Mixed lineage leukemia histone methylases (MLLs)
are human histone H3 lysine 4 (H3K4)-specific histone
methyltransferases (HMTs) that play critical roles in

tionally regulated by E2, and that MLLs, in coordina-
tion with ERs, regulate E2-induced activation of
HOXC13.
Results
HOXC13 is transcriptionally regulated by E2
ERs are major players in E2-mediated regulation of
E2-responsive genes [41,42]. In general, upon binding
to E2, ERs are activated. The activated ERs bind to
E2 response elements (EREs) present in the promoter
of E2-responsive genes, leading to their transcriptional
activation [43]. In this work, before examining the
E2-mediated regulation of HOXC13, we analyzed its
promoter for the presence of any EREs. Our results
demonstrated that the HOXC13 promoter contains six
putative EREs (ERE1 ⁄ 2 sites) within )1to)3000 bp
upstream of the transcription start site (Fig. 1). All of
the EREs show 100% homology with ERE1 ⁄ 2 sites
(GGTCA or TGACC) but not with the consensus full
ERE sequence (GGTCAnnnTGACC). The presence of
these EREs in close proximity to the transcription start
site indicated that HOXC13 might be potentially regu-
lated by E2 via the involvement of ERs.
In order to examine whether HOXC13 is regulated
by E2, we treated a steroidogenic human cell line
(JAR cells, of choriocarcinoma placental origin, cul-
tured in phenol-red free medium containing activated
charcoal-treated fetal bovine serum) with different
concentrations (1–1000 nm) of E2 for 8 h. The RNA
was isolated from the E2-treated cells and analyzed by
RT-PCR, using HOXC13-specific primers (Fig. 2;

3
E
R
E
2
E
R
E
1
+
1
–
2
3
4
E
R
E
6
E
R
E
5
E
R
E
4
E
R
E

E2-induced activation of HOXC13, we knocked down
ERa and ERb separately, using specific antisense oli-
gonucleotides, in JAR cells and exposed the cells to
100 nm E2 for an additional 8 h. A scramble antisense
oligonucleotide (with no homology to ERs) was used
as a negative control. Our results demonstrated that
application of ERa or ERb antisense oligonucleotide
knocked down the respective genes efficiently, at both
the mRNA and the protein level (Fig. 3A,B, lanes 4–6,
and data not shown; the quantifications are shown in
the respective bottom panels). After confirming effec-
tive knockdown, we analyzed the RNA from these ER
knockdown and E2-treated cells for the expression lev-
els of HOXC13 using RT-PCR. As seen in Fig. 3A,B,
HOXC13 expression was increased upon exposure to
E2 (compare lanes 1 and 2), and application of scram-
ble antisense oligonucleotide did not have any signifi-
cant effect on E2-mediated activation of HOXC13.
Interestingly, upon knockdown of either ERa or ERb,
the E2-dependent activation of HOXC13 was sup-
pressed almost to the basal level (Fig. 3 A,B, compare
lanes 5 and 6 with lanes 1 and 2; quantifications are
shown in the respective bottom panels). These results
demonstrated that both ERa and ERb are essential for
E2-mediated transcriptional activation of HOXC13.
MLLs play critical roles in E2-induced HOXC13
expression
As MLLs are well known as master regulators of
HOX genes, and several MLLs are implicated in E2
signaling, we examined whether any of the MLLs are

(relative to actin)
0
0.2
0.4
0.6
0.8
02468121624
Time (h)
HOXC13 expression
(relative to actin)
[E2] (n
M
)
0
0.2
0.4
0.6
0.8
0
0.1
1
10
100
1000
A
B
C
D
Fig. 2. Effect of E2 on HOXC13 gene expression. (A, B) JAR cells
were initially grown in phenol red-free medium, and treated with

ished the E2-mediated activation of HOXC13
(Fig. 4C). These results demonstrated that the MLL
family of HMTs, especially MLL3, play critical roles
in the E2-mediated regulation of HOXC13.
E2-induced recruitment of ERs and MLLs in the
HOXC13 promoter
As the HOXC13 promoter contains several ERE1 ⁄ 2
regions within the first 3000 nucleotides upstream of
the transcription start site, we analyzed the involve-
ment of some of these EREs (ERE1–ERE4, located at
)234, )1260, )1788 and )2000 bp upstream) by ana-
lyzing the in vivo binding of ERs and MLLs. We ana-
lyzed the in vivo binding of the different factors in the
absence and presence of E2, using chromatin immuno-
precipitation (ChIP) assays [34], using antibodies
against ERs and MLLs. ChIP experiments were also
performed in parallel with the use of antibody against
actin as a nonspecific negative control. In brief, JAR
cells were treated with 100 nm E2 for 6 h, and control
and E2-treated cells were then subjected to ChIP anal-
ysis. The immunoprecipitated DNA fragments were
PCR amplified using primers specific for ERE1, ERE2,
ERE3 and ERE4 of the HOXC13 promoter. As seen
in Fig. 5A, no significant binding of actin was
observed in any of the EREs, irrespective of the
absence and presence of E2. Binding of ERa and ERb
was increased in both ERE1 and ERE2 of the
HOXC13 promoter (Fig. 5A, lanes 1–4). The levels of
E2-induced binding of ERa and ERb were higher in
ERE2 than in ERE1. ERE3 and ERE4 were not sensi-

a
ERb antisense GAATGTCATAGCTGA
a
MLL1 antisense TGCCAGTCGTTCCTCTCCAC
a
MLL2 antisense ACTCTGCCACTTCCCGCTCA
a
MLL3 antisense CCATCTGTTCCTTCCACTCCC
a
MLL4 antisense CCTTCTCTTCTCCCTCCTTGT
a
Scramble antisense CGTTTGTCCCTCCAGCATCT
a
a
Phosphorothioate antisense oligonucleotide.
K. I. Ansari et al. Estrogen-mediated HOXC13 activation involving MLL
FEBS Journal 276 (2009) 7400–7411 ª 2009 The Authors Journal compilation ª 2009 FEBS 7403
which are close to the transcription start site, are
mostly responsible for E2-dependent binding of ERs
and MLLs and hence the regulation of HOXC13.
ERE2 appeared to have more critical roles (sensitivity
to E2) than the other EREs examined. ERE3 and
ERE4, which are located far upstream ()1788 bp or
further), were not sensitive to E2-dependent binding of
any of the MLLs⁄ ERs, indicating no significant roles
of these EREs in HOXC13 activation (Fig. 5A).
To further confirm the E2-dependent binding of
ERs and MLLs to the HOXC13 promoter, we
analyzed their binding profiles in a time-dependent
manner in ERE1 and ERE2 (Fig. 5B). In agreement

trimethylation level in ERE1 and ERE2. Our results
demonstrated that in both ERE1 and ERE2, the levels
of RNAPII and H3K4-trimethylation were increased in
the presence of E2 (Fig. 5B). These results demon-
strated that both ERE1 and ERE2 (especially ERE2)
coordinate the binding of ER and MLL coregulators as
well as RNAPII, and regulate the E2-mediated tran-
scriptional activation of HOXC13. It is important to
note that although ERE2 is located far upstream
(1260 bp away from the transcription start site), we still
observed significant transcription-dependent increases
in RNAPII binding to these EREs. These observations
suggest that there is probably a looping of the large
promoter regions so that far upstream cis-elements
could be placed closer to the promoter proximal sites
Scramble ERβ
β-actin
HOXC13
ERβ
E2 (100 n
M)
Antisense
(µg)
Expression
(relative to actin)
0.0
0.2
0.4
0.6
0.8

HOXC13
Fig. 3. Effect of depletion of ERa and ERb on E2-induced expres-
sion of HOXC13. JAR cells were grown up to 60% confluency prior
to treatment with different concentrations of ERa-specific and
ERb-specific phosphorothioate oligonucleotides by using ifect
transfection (MoleculA). Control cells were treated with a scramble
antisense oligonucleotide with no homology with the ERa and ERb
genes. The antisense oligonucleotide-transfected cells were incu-
bated for 24 h and then treated with E2 (100 n
M) for an additional
8 h. Cells were harvested and subjected to RNA preparation. The
mRNA was subjected to RT-PCR analysis by using primers specific
for HOXC13 along with ERa and ERb. b-Actin was used as control.
The RT-PCR products were analyzed in agarose gel. Quantification
of transcript accumulation on the basis of RT-PCR products
(average of three replicates) is shown beneath the respective gel
image. Bars indicate standard errors. Values were assumed to be
significantly different at P £ 0.05. The results of experiments involv-
ing ERa and ERb are shown in (A) and (B), respectively.
Estrogen-mediated HOXC13 activation involving MLL K. I. Ansari et al.
7404 FEBS Journal 276 (2009) 7400–7411 ª 2009 The Authors Journal compilation ª 2009 FEBS
and coordinate with RNAPII and other transcription
factors during transcription initiation [45,46].
In addition, binding of some MLLs to certain EREs
even prior to the addition of E2 suggests that this
binding might be linked to the basal transcriptional
regulation of the gene. Furthermore, we also observed
that the recruitment of MLL2 is induced by E2 at both
ERE1 and ERE2. However, the recruitment of other
MLLs (i.e. MLL1, MLL3, and MLL4) at ERE1 is not

E2 (100 n
M) – + +
Antisense
1 2 3
S
c
r
a
m
b
l
e
β-actin
MLL3
M
L
L
3
HOXC13
Antisense
S
c
r
a
m
b
l
e
β-actin
MLL3

L
L
4
HOXC13
Antisense
S
c
r
a
m
b
l
e
β-actin
MLL4
M
L
L
4
Expression
(relative to actin)
0.0
0.4
0.8
1.2
MLL2
HOXC13
B
HOXC13
E2 (100 n

L
L
2
Expression
(relative to actin)
0.0
0.4
0.8
1.2
MLL1
HOXC13
HOXC13
123
1 2 3
123
1 2 3
123
1 2 3
123
–E2 (100 nM)+ +
1 2 3
S
c
r
a
m
b
l
e
β-actin

assumed to be significantly different at
P £ 0.05.
K. I. Ansari et al. Estrogen-mediated HOXC13 activation involving MLL
FEBS Journal 276 (2009) 7400–7411 ª 2009 The Authors Journal compilation ª 2009 FEBS 7405
binding of promoters. This binding may be critical
for regulation of basal transcription of the target
genes. On the other hand, MLLs might be recruited
to the HOXC13 promoter via protein–protein interac-
tions (direct or indirect) with ERs. Amino acid
sequence analysis demonstrated that MLL1–MLL4
have LXXLL domains [also called nuclear receptor
(NR) boxes], which are known to be involved in
E2-dependent interactions with ERs [12]. MLL1 has
only one LXXLL domain, whereas MLL2, MLL3
and MLL4 have multiple LXXLL domains [12]. In
fact, MLL2, MLL3 and MLL4 have recently been
shown to interact with ERs, and are involved in
the E2-mediated activation of E2-responsive genes
[12,35–38]. In the present study, we examined whether
all of the MLLs that are involved in the E2-mediated
activation of HOXC13 directly bind to the EREs, or
whether they are recruited to EREs via interactions
with ERs in an E2-dependent manner. To examine
this, we knocked down both ERa and ERb sepa-
rately, exposed the cell to 100 nm E2 for 6 h, and
analyzed the status of the binding of all the MLLs to
ERE1 and ERE2 of the HOXC13 promoter (Fig. 6).
As expected, our results demonstrated that binding of
each of the MLLs (MLL1–MLL4) was increased in
ERE2 of the HOXC13 promoter in the presence of

MLL3
H3K4-tri
Met
RNAPII
MLL2
MLL4
Time (h) 0 ¼ ½ 1 2 4 6 8 0 ¼ ½ 1 2 4 6 8
Fig. 5. E2-dependent recruitment of ERa,
ERb and MLLs (MLL1–MLL4) in ERE1,
ERE2, ERE3 and ERE4 of the HOXC13
promoter. (A) E2-treated (100 n
M for 6 h)
and untreated JAR cells were subjected
toChIP assay, using antibodies against ERa,
ERb MLL1, MLL2, MLL3, and MLL4.
b-Actin antibody was used as control IgG.
The immunoprecipitated DNA fragments
were PCR amplified using primers specific
for ERE1, ERE2, ERE3 and ERE4 of the
HOXC13 promoter. (B) Dynamics of recruit-
ment of ERa,ERb and MLLs (MLL1–MLL4),
H3K4-trimethyl and RNAPII into ERE1 and
ERE2 of the HOXC13 promoter under E2
treatment using ChIP assays. JAR cells
were treated with 100 n
M E2 for different
time periods (0–8 h), and then subjected to
ChIP assay using different antibodies.
Immunoprecipitated DNA fragments were
PCR amplified using primers specific for

factors that regulate critical genes that are necessary
for cell differentiation and development [1,2]. Despite
their critical and well-characterized functions, the regu-
latory mechanisms that drive HOX gene expression are
mostly unknown. Although the mechanism is unclear,
several hormones have recently been shown to regulate
HOX gene expression, and the endocrine regulation of
HOX genes appears to allow the generation of struc-
tural and functional diversity in both developing and
adult tissues [47].
HOXC13 is a homeobox-containing gene that plays
critical roles in hair development. Hair follicle develop-
ment, male-specific and female-specific hair patterning
and sexual differentiation are strongly dependent on
steroid hormones such as E2, progesterone, and andro-
gens [3–5,10]. Herein, we have demonstrated that the
HOXC13 gene is transcriptionally regulated by E2.
ERa and ERb are two major players in E2-dependent
gene activation [41]. Our studies demonstrated that
antisense oligonucleotide-mediated knockdown of
either ERa or ERb downregulated the E2-mediated
activation of HOXC13, indicating their critical roles in
the process. ER-mediated regulation of E2-sensitive
genes is a complicated process [43]. In the presence
of E2, ERs are activated and bind to the EREs of
Antisense
A
B
Input
MLL1

l
e
E
R
E
R
Input
MLL1
– + + + – + + +
MLL2
MLL3
MLL4
N
o
ne
S
c
r
a
m
b
l
e
E
Rα
E
Rβ
N
one
S

ERE2 of the HOXC13 promoter. (B) Interac-
tion of MLL3 with ERs. JAR cells were
treated with 100 n
M E2 for 6 h before being
harvested for preparation of nuclear extract.
The extracts were immunoprecipitated
by using MLL3 antibody. The immuno-
precipitated MLL3 complexes were then
analyzed by western blot, using ERa and
ERb antibodies. Immunoprecipitation with
protein G agarose beads was used as
negative control.
K. I. Ansari et al. Estrogen-mediated HOXC13 activation involving MLL
FEBS Journal 276 (2009) 7400–7411 ª 2009 The Authors Journal compilation ª 2009 FEBS 7407
E2-responsive genes, eventually resulting in transcrip-
tion activation [41]. In addition to ERs, E2-mediated
gene activation requires various other coregulators and
coactivators that result in chromatin modification and
remodeling [40,48]. Our results described herein dem-
onstrated that MLLs and ERs play crucial roles in the
E2-mediated regulation of HOXC13. Knockdown of
MLLs (especially MLL3) suppressed the E2-mediated
activation of HOXC13.
In general, ERs, along with various coregulators,
are recruited to EREs present in the promoters of
E2-responsive genes [41]. Our sequence analysis
demonstrated that the HOXC13 promoter contains at
least six EREs within )3000 bp upstream of the tran-
scription start site. In vivo binding analysis (ChIP)
demonstrated that, in the presence of E2, ERs bind

HOXC13 expression, we expected (as observed; Fig. 5)
them to bind to HOXC13 EREs as a function of E2.
However, irrespective of the relative importance of the
MLLs (MLL1–MLL4), ChIP analysis (Fig. 5) showed
efficient E2-dependent binding of all the MLLs
in ERE2. It should be noted that the ChIP assay
does not provide a truly quantitative measurement in
terms of activity of the enzyme, although it provides
important information about relative binding
efficiency. This might explain the difference in MLL
binding profile (ChIP data) versus their activity in
knockdown experiments.
Our studies demonstrated that, in addition to
MLL2–MLL4, MLL1 is also recruited to ERE2 of
the HOXC13 promoter in an E2-dependent manner.
Amino acid sequence analysis demonstrated that each
MLL (MLL1–MLL4) contains one or more LXXLL
domains (NR boxes), which are known to interact
with nuclear receptors (NRs) and mediate ligand-
dependent gene activation [12]. MLL1 contains one
NR box, whereas MLL2–MLL4 contain several (three
to four) NR boxes, indicating that each of the MLLs
has the potential to interact with ERs and be
involved in E2-mediated gene activation [12].
Although further studies are needed to understand
the detailed roles of different MLLs and their coordi-
nation with ERs, our studies have demonstrated that
MLL1–MLL4 are involved in E2-mediated HOXC13
regulation. Furthermore, the E2-dependent increase in
histone H3K4-trimethylation level suggested that

Estrogen-mediated HOXC13 activation involving MLL K. I. Ansari et al.
7408 FEBS Journal 276 (2009) 7400–7411 ª 2009 The Authors Journal compilation ª 2009 FEBS
Experimental procedures
Cell culture, E2 treatment, and antisense
oligonucleotide experiments
Human choriocarcinoma placenta (JAR) cells obtained from
the ATCC were maintained in DMEM (Sigma, St Louis,
MO, USA) supplemented with 10% fetal bovine serum,
2mml-glutamine and penicillin ⁄ streptomycin (100 units
and 0.1 mgÆmL
)1
, respectively) in a humidified CO
2
incu-
bator, as described previously [11,50,51]. Prior to E2 treat-
ment, JAR cells were grown in phenol red-free DMEM-F12
(Sigma), containing 10% activated charcoal-stripped fetal
bovine serum for at least three generations. The final round
of the cells were grown up to 70% confluency and treated
with different concentrations (0–1000 nm) of E2 for varying
time periods. The cells were then harvested and subjected to
either RNA and protein extraction or ChIP assay.
For treatment of JAR cells with antisense oligonucleo-
tides, cells were grown up to 60% confluency in 60 mm cul-
ture plates and transfected with varying amounts (3–9 lg)
of different antisense oligonucleotides. Briefly, cocktails of
different amounts of antisense oligonucleotide and transfec-
tion reagents (ifect, MoleculA) were made in the presence
of 300 lL of culture medium (without supplements) by
incubating for 30 min, as instructed by the manufacturer.

The first cDNA was synthesized in a 25 lL reaction volume
containing 500 ng of RNA, 2.4 lm oligo(dT) (Promega,
Madison, WI, USA), 100 units of Moloney murine
leukemia virus reverse transcriptase, 1· first-strand buffer
(Promega), 100 lm each of dATP, dGTP, dCTP, and dTTP
(Invitrogen, Carlsbad, CA, USA), 1 mm dithiothreitol, and
20 units of RNaseOut (Invitrogen). The cDNA was diluted
to 100 lL, and 5 lL of the diluted cDNA was used for
PCR performed with the gene-specific primer pairs
described in Table 1. The PCR program consisted of 30
cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s,
with a final extension at 72 °C for 5 min. Each of the
experiments was repeated three times. The normality of the
data was analyzed by using t-tests, and ANOVAs were
performed at a 5% level of significance.
For western blot analysis, 25 lg of protein extract was
subjected to SDS⁄ PAGE and transferred to nitrocellulose
membranes. The membranes were then probed with anti-
bodies against MLL1 (Bethyl laboratory), MLL2 (Bethyl
laboratory), MLL3 (Abgent, San Diego, CA, USA), MLL4
(Sigma), ERa (Santa Cruz Biotechnology, Santa Cruz, CA,
USA), ERb (Santa Cruz), and b-actin (Sigma), and devel-
oped using the alkaline phosphatase method.
ChIP assays
ChIP assays were performed by using an EZ Chip chroma-
tin immunoprecipitation kit (Upstate, Billerica, MA, USA),
as described previously [34]. In brief, cells were fixed in 4%
formaldehyde, lysed, and sonicated to shear the chromatin.
The fragmented chromatins were precleaned with protein G
agarose and subjected to overnight immunoprecipitation

We thank S. Mandal, B. P. Mishra and other labora-
tory members for helpful discussions. This work was
supported in part by ARP (00365-0009-2006) and the
American Heart Association (0765160Y).
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